This invention concerns an improved method for the determination of lipid bound sialic acid in plasma or serum which is less expensive, less time consuming, less variable from sample to sample, and less dependent upon the skill and experience of the person performing the test. Additionally this invention concerns an improved test kit which contains a unique standard which will allow greater reproducibility of test results and more consistency throughout the world.
Much work has been done which indicates that elevated sialic acid content in blood sera of a patient is an indication of the presence of cancer. For example, U.S. Pat. No. 4,146,603 to Davidson, et al. discloses and claims a fairly complex series of procedures whereby elevated sialic acid content is a determinant with respect to cancer specific determinations.
MacBeth and Bekesi, Cancer Res. 22:1170-1176 (1962) measured plasma glycoproteins and found galactose and mannose values were seen in breast cases without metastases. Kloppel, et al., Proc. Natl. Acad. Sc. 74:3011-3013 (1977) reported 2.5-fold increases of serum sialic acid glycolipids in mice bearing transplantable mammory carcinomas and 2-fold increases in human carcinoma patients. Kloppel, et al., Am. J. Vet. Res. 39:1377-1380 (1978) also reported increases of sialic acid in 93% of 24 dogs. In leukemia AKR/J mice, Lengle, J. Natl. Cancer Inst. 62:1565-1567 (1979) found increased lipid bound sialic acid in their plasma and thymic lymphocytes. Lipid bound sialic acid levels were found increased in plasma and erythrocytes of humans bearing melanomas. Portouklian, et al., Biochem. Biophys. Res. Commun. 85:916-920 (1978). Chromatographic separation and purification on columns was followed by evaluation on chromatoplates. Silver, et al., Cancer 41:1497-1499 (1978); Cancer Res. 39:5036-5042 (1979) have reported elevated serum sialic acid values in melanoma patients that were significantly related to the tumor burden. However, 36% of patients with observable tumors showed no elevated serum sialic acid. Hogan-Ryan, et al., Br. J. Cancer 41:587-592 (1980) reporting on total bound serum sialic acid in patients with breast cancer found elevations that corresponded with tumor stage.
One specific method over which the present invention is an improvement is disclosed in the American Association for Cancer Research Annual Meeting PROCEEDINGS Vol 21, March 1980 as Abstract No. 728 by Katopodis, et al. This method requires that a 100 ul plasma sample (reduced to 50 ul) be extracted with 6 ml of a chloroform/methanol mixture, (2 to 1, volume to volume ratio). The lipid extract is then partitioned with 0.2 of its volume of water. The aqueous phase is evaporated to dryness and the residue redissolved in water. The lipid bound sialic acid is then purified by trichloroacetic acid-phosphotungstic acid precipitation and, after the removal of the supernatant from the resultant precipitate, the precipitate is determined by the Svennerholm and Miettien method (Svennerholm, Quantitative Estimation of Sialic Acid . . . , Biochem. Biophys, Acta. 24, pp. 604-611 (1957)).
Another specific method over which the present invention is an improvement is disclosed in Katopodis and Stock, U.S. Pat. No. 4,342,567, issued Aug. 3, 1982. This method is similar to the foregoing but requires only about 50 ul of sample rather than the 100 ml required by the prior art method. The drying step is eliminated and there is no use of trichloracetic acid. Phosphotungstic acid is used alone.
This method suffers from a number of disadvantages including the following: the need for a precisely defined 44.7 ul starting sample; lipid bound sialic acid is lost during the tube inversion step creating reduced final values; precipitation of the lipid bound sialic acid with phosphotungstic acid is not complete, which is a particular problem when working with samples in which the amount exceeds normal values by only small amounts (e.g. early in cancer development); the rapidity of the test is limited by the 5 minutes waiting time after phosphotungstic acid addition and the cost of the test is not as low as is desirable. Another method over which the present invention is an improvement is disclosed in Katopodis, U.S. Pat. No. 4,748,128, issued May 31, 1988. This prior method consists of the following steps:
(a) diluting a predetermined volume of a blood plasma or serum with distilled water to a volume about four times that of the predetermined volume; PA1 (b) mixing the diluted sample for a suitable period of time to obtain a substantially homogeneous sample; PA1 (c) cooling the mixed, diluted sample to about 0 degrees to 5 degrees C.; PA1 (d) adding to the cooled sample a mixture of a chlorinated lower alkyl hydrocarbon and a lower alkyl alcohol, the volume of the mixture added being about sixty times the predetermined volume of the blood plasma or serum sample, and the volume ratio of chlorinated hydrocarbon to alcohol in the mixture being about 2:1 and its temperature about 0 degrees to 5 degrees C.; PA1 (e) mixing the resulting admixture for a suitable period of time to dissolve matter present in the sample in the chlorinated hydrocarbon/alcohol mixture; PA1 (f) diluting the admixture with deionized distilled water at a temperature from about 0 degrees to 5 degrees C., the volume added being about ten times the predetermined volume of the blood plasma or serum sample; PA1 (g) treating the diluted admixture for a suitable period of time to permit formation of a substantially clear upper phase; PA1 (h) separately recovering from the clear upper phase so formed a predetermined volume of the upper phase; PA1 (i) adding to the predetermined volume of the upper phase an amount of a mixture of a protein-precipitating agent and an adsorbing material, the amount of mixture being effective to cause precipitation of the lipid bound sialic acid and to adsorb the precipitated lipid bound sialic acid; PA1 (j) mixing the resulting admixture; PA1 (k) separately recovering the resulting adsorbed precipitate; PA1 (l) suspending the precipitate in a suitable volume distilled water; and PA1 (m) adding to the suspended precipitate a volume of resorcinol reagent, mixing, boiling for 15 minutes, cooling for 10 minutes in an ice bath, centrifuging, adding a mixture of butylacetate and n-butanol (85:15 v/v) in a volume about twice the volume of the resorcinol reagent, mixing, centrifuging, separating the organic layer, reading at 580 nm the extracted blue color present in the organic layer, and determining the amount of lipid bound sialic acid by comparing the optical density reading obtained at 580 nm to a standard curve developed from a known sample of n-acetyl neuraminic acid (NANA) under the same conditions and applying the formula: EQU LSA (mg/100 ml plasma)=(x.multidot.10.sup.5 ul)/y.multidot.z ul.multidot.1000) PA1 (a) diluting a predetermined volume of a blood plasma or serum sample with buffer solution to a volume about five (5) times that of the predetermined volume; PA1 (b) mixing the diluted sample for a suitable period of time to obtain a substantially homogeneous sample; PA1 (c) adding to the sample a mixture of a chlorinated lower alkyl hydrocarbon and a lower alkyl alcohol, the volume of the mixture added being about twenty (20) times the predetermined volume of the blood plasma or serum sample, and the volume ratio of chlorinated hydrocarbon to alcohol in the mixture being about 1:1; PA1 (d) mixing the resulting admixture for a suitable period of time to dissolve matter present in the sample in the chlorinated hydrocarbon/alcohol/buffer mixture; PA1 (e) centrifuging the admixture for a suitable period of time at a suitable speed to permit formation of a substantially clear upper phase; PA1 (f) separately recovering from the clear upper phase so formed a predetermined volume of the upper phase; PA1 (g) adding to the predetermined volume of the clear upper phase a color development reagent; PA1 (h) mixing the resulting admixture; PA1 (i) boiling the admixture; PA1 (j) cooling the admixture; PA1 (k) mixing, then centrifuging said admixture, separating the clear supernatant from the precipitated material; and PA1 (l) determining the amount of lipid bound sialic acid present in the supernatant and thereby the amount present in the blood plasma or serum sample.
where x=NANA read from the standard curve, y=the volume of the upper phase recovered divided by the total volume of the entire upper phase and z=the predetermined volume of the blood plasma or serum sample.
The present invention provides an improved method for determining the amount of lipid bound sialic acid present in a sample of plasma or serum. The present invention is simpler, more economical, faster, more easily automated, and is suitable for the preparation of monoclonal antibodies due to its purity. It requires less chemical reagents and is superior in terms of specificity and sensitivity than the methods of the prior art. The procedure of the present invention differs significantly from known methods in that the present invention eliminates the need to use a protein precipitating agent and eliminates the need to use a special standard. The present invention reads the optical density of standard commercially available n-acetyl neuraminic acid (NANA) and thus eliminates the need to construct a standard curve. This enhances reproducibility from laboratory to laboratory and enables the user to more quickly and more accurately determine the level of sialic acid in the sample.
The prior art requires the use of a protein precipitating agent such as phosphotungstic acid and an adsorbing material to cause the precipitation of the lipid bound sialic acid. The present invention does not require this step as it has been found that it is unnecessary where the ratio of the chlorinated hydrocarbon to alcohol is approximately 1:1 in the buffered solution.
Significantly the present invention provides an improved procedure for determining the concentration of lipid bound sialic acid in a sample of human blood plasma or serum by comparing the sample with a standard consisting of n-acetyl neuraminic acid (NANA) in commercially available form rather than human or animal blood plasma. Commercially available NANA has the advantage of being readily available, is extremely stable and has a long shelf life. It also eliminates the need to construct a standard curve for the standard as in the prior art. It is also superior to the methods of the prior art since the elimination of the protein precipitating reagent enables the LSA fraction to be used easily for the preparation of specific monoclonal antibodies.
Finally, the chemistry of the present invention eliminates the need to use a mixture of butylacetate and n-butanol as in the prior art. In addition to the making the test less expensive, it avoids the need to work with this mixture which has a foul smell, causes a burning sensation in the nasal membrane of the technician and is generally difficult to work with.